The long-term goal of this research is to determine the epidemic potential of California serogroup (CAL) bunyavirus evolutionary events. Unprecedented numbers of CAL virus central nervous system (CNS) infections recently have been diagnosed in Russia, and reassortant CAL viruses have been isolated. Whether the increased incidence of these CNS infections is attributable to newly emerged bunyaviruses, to bunyavirus evolutionary events, to vector-associated phenomena, or to other factors will be investigated. Because the medically-important CAL viruses and their vectors are holarctic in distribution, our studies will provide critical information about the epidemiologic consequences of CAL virus evolution in North America as well as in Russia. The evolutionary potential of CAL viruses from Russia and North America will be determined in natural and alternate vector species. The ability of selected CAL viruses to reassort their RNA segments will be determined. Vector and viral phylogenetic constraints on reassortment potential will be determined. Intramolecular evolution will be investigated by direct amplification of virus sequences from infected mosquitoes; polymerase chain reaction (PCR) will be used to amplify target viral genomic sequences from mosquitoes, denaturing gradient gel electrophoresis used to identity candidates for sequencing, and sequence analyses used to define differences between isolates. These studies will provide information concerning the role of the arbovirus cycle in promoting viral genomic stability or plasticity and the role of vector passage in maintaining arbovirus cycle integrity and transmission potential. Specific molecular probes, PCR protocols, and immunologic techniques will be developed for rapid genotypic identification and diagnosis of CAL viruses. Many of the vector species associated with holarctic transmission of CAL viruses are members of species complexes and difficult to identity by conventional taxonomic criteria. Molecular techniques will be developed to identity specifically the vector species involved in virus transmission. Relationships between viral genotypes, vector species, and disease incidence in Russia will also be elucidated. CAL viruses previously isolated or newly isolated and vectors from premises where human CNS infections have occurred will be genotypically, phenotypically, and biologically characterized, the latter in terms of vector and vertebrate host interactions. These studies will provide information on the distribution of CAL viruses and relationships between viral genotypes, vector species, and CNS disease incidence. Such knowledge will be essential for eventual development of effective surveillance, diagnosis, and control strategies for these important pathogens of humans.